Abstract- This R01 renewal seeks to continue the PI's creative, productive, and significant research program, which has enjoyed uninterrupted NIH funding since 1996, focused on lipid/lipoprotein metabolism in the pathogenesis of cardiovascular disease (CVD) and cardiometabolic disease (CMD). For over 15 years, the PI's research program has focused on ATP binding cassette transporter A1 (Abca1) and lipid and lipoprotein metabolism using tissue/cell-specific Abca1 knockout mice. Studies in hepatocyte-specific Abca1 knockout (HSKO) mice provided novel mechanistic insights into how hepatic Abca1 impacts the production and catabolism of all three major classes of plasma lipoproteins (VLDL, LDL, HDL) that contribute to CVD and CMD. Despite the massive (~80%) drop in plasma HDL relative to control mice, hyperlipidemic HSKO mice maintained macrophage reverse cholesterol transport and had decreased aortic root atherosclerosis compared to controls. HSKO mice also had increased plasma HDL cholesterol transport into the feces, decreased hepatic insulin signaling and de novo lipogenesis (DNL), increased mitochondrial respiration, increased LDL receptor expression, and increased hepatic VLDL triglyceride secretion. This unique phenotype was associated with diminished antegrade trafficking of lysosomal free cholesterol (FC) to the plasma membrane of hepatocytes, with no differences in total hepatic lipid mass between HSKO and control mice. Based on this work, our global hypothesis is that targeted deletion of hepatocyte Abca1 enhances hepatocyte retrograde FC redistribution from the plasma membrane to intracellular compartments, reprogramming insulin-mediated anabolism towards a coordinated catabolic program that reduces hepatic DNL, improves mitochondrial metabolism, and increases TG secretion, thereby decreasing hepatic steatosis when mice are stressed with a high-fat diet. This metabolic reprogramming in the absence of hepatic Abca1 results in a novel form of selective insulin resistance in HSKO mice, in which hepatic DNL is suppressed, but gluconeogenesis is appropriately downregulated by insulin. Future studies will address gaps in knowledge and barriers to progress, including: Specific aim 1) how Abca1 deletion alters FC distribution (or trafficking) among the plasma membrane, endoplasmic reticulum, and other organelles; Specific aim 2a) how insulin signaling and endoplasmic reticulum FC interact to activate Srebp1c; and Specific aim 2b) whether loss of Abca1 leads to increased mitochondria FC and/or lysophospholipid that improves mitochondrial respiration. Our proposed studies will build a more comprehensive and expansive model for Abca1 as a central control point of metabolic homeostasis and propel our previous 5-year discoveries in new directions, thereby advancing the field. Impact- Our studies will continue generating new discoveries regarding regulation of hepatic lipogenesis and the emerging role of Abca1 and FC trafficking in reprogramming hepatic lipid metabolism, a key contributor to CVD and CMD.